Cultivation and Screening of Microalgae Isolates for Anti-Tumour Bioactive Compounds

Chin Wei LuLim Wei Xin Fiona SUPERVISOR: Dr. New Jen YanCO-SUPERVISOR: Dr. Charmaine Lloyd

Microalgae Microalgae are photoautotrophic, unicellular

algae cells

Being explored as alternative sources, over terrestrial plants, of high-value products, such as renewable biofuels and nutritional supplements (e.g. dietary anti-oxidants, vitamins)

Great diversity

Simple growth requirements

High growth efficiencies

Mass cultivation offshore

Introduction

Lugol’s iodine-stained microalgae isolated from local water bodies

1

Nitrogen-deficient cultivation increased lipid content in Chlorella vulgaris microalgae from 14.5% to 24.6% of dry weight (Mujtaba et al., 2012)

Ultraviolet-A irradiation increased both lipid content and degree of unsaturation, in Nitzschia closterium (Bacillariophyceae) and Isochrysis zhangjiangensis (Chrysophyceae) microalgae (Huang and Cheung., 2011)

Introduction

2

Flexibility to alter biomass composition

Introduction

3

Anti-cancer activity in

macroalgae

Reduction in tumour size of Agrobacterium tumefaciens-infected potato discs, after treatment with ethanol crude extracts from Jania rubens algae (Ibrahim et al., 2005)

Jania rubens red algae

Pepsin-digested extracts from Caulerpa microphysa algae induced tumour shrinkage in immunocompromised mice transplanted with human leukaemia cell lines (Lin et al., 2012)

Caulerpa microphysa algae

http://www.umema.it/Alghe/album/Rosse/slides/02%20Jania%20Rubens.html

http://biogeodb.stri.si.edu/pacificalgae/specie/19

I. To determine if crude extracts from local water bodies-originated microalgae species (vs38, vs88, vs31 and KK6) have anti-tumour effects against cancerous basophils KU812 and oestrogen receptor-negative breast cancer cell line MB231

II. To compare the efficacy of these crude extracts with anti-cancer antibiotic Actinomycin-D in inhibiting KU812 and MB231 proliferation

Objectives

4

Cultivation of microalgae strains• 4 morphologically-distinct

microalgae strains isolated from local water bodies

• Incubated in an enclosed room at (25±1)⁰C with illumination

• Half of the respective algal cultures were irradiated under UV-C for 4 hours (with swirling every 10 minutes) -> 24 hours recovery timevs38

KK6

vs88

vs31

Materials and Methods

5

Harvesting of microalgae strains Washing -> Lyophilisation -> Solvent

extraction (Hexane & Ethanol & dH2O)-> Ultra-sonication -> Vortex with micro glass beads -> Incubation -> Evaporate to dryness

Mass of dried extracts were recorded

Re-dissolve dried extracts in DMSO to achieve a Master stock of microalgae extract(2000mg/mL)

Materials and Methods

Microtube

Glass tube

2000mg/mL6

Anti-tumour test for microalgae crude extracts and Actinomycin-D

1. Cancer cells were seeded at a density of 2.0x105 living cells/mL in each well on a 96-well plate, in replicates of five for each working concentration

2. Cells then incubated at 37◦C, 5% CO2 for 24 hours to acclimatise/adhere to substratum

3. Cells then added with microalgae crude extract master stocks, to the following working concentrations:

o 2.0mg/mLo 1.0mg/mLo 0.5mg/mLo 0.25mg/mLo 0.0625mg/mL

Materials and Methods

7

4. Treated cells then incubated at 37◦C, 5% CO2 for 48 hours

5. Cell viability at each microalgae crude extract working concentration assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Assay

Anti-tumour test for microalgae crude extracts and Actinomycin-D

Statistical analysis Significant differences in cell viability between

samples were tested for using Student’s t-test for non-paired samples and Mann-Whitney-U test

Materials and Methods

8

Negative controlCancer cells cultured in pure culture medium only

Safe DMSO controlCancer cells cultured in 0.1% (v/v) DMSO

MTT controlCancer cells cultured in 50% (v/v) DMSO

Materials and Methods

9

Analysis of results Growth inhibitory or stimulatory effects of microalgae crude

extracts assessed only at 0.25mg/mL working concentration

Growth stimulatory effect population of living cancer cells in microalgae test is significantly larger than in negative control

Growth inhibitory effect population of living cancer cells in microalgae test is significantly smaller than in negative control

Materials and Methods

10

Growth inhibition efficiency =

[(cell viability in negative control – cell viability in microalgae test)/cell viability in negative control] x 100%

MB231 cell viability in stressed and non-stressed microalgae vs88 distilled water crude extract tests, and in their respective negative controls, after 48-hour exposure (representative bar chart)

Materials and Methods

11

Viable breast cancer MB231 cells (after MTT assay) seen under microscope. LEFT: incubation with inhibitory microalgae crude extract. RIGHT: negative control

Results and Discussion

Effects of test microalga on cancerous basophils KU812, BY STRAIN

Y-axis = Population of living cancer cells presentBlue – test microalgae crude extractPale blue – negative control 12

Results and DiscussionEffects of test microalga on breast cancer cell line MB231, BY STRAIN

Dark grey – test microalgae crude extractPale grey – negative controlY-axis = Population of living cancer cells present 13

Growth of cancerous basophils KU812 is significantly inhibited by microalgae strain vs31, but not by vs38, vs88 and KK6.

Growth of breast cancer cell line MB231 is significantly inhibited by microalgae strains vs88 and vs38, but not by vs31 and KK6.

1) Microalgae strains vs31 and vs88 are promising candidates for further exploration into their anti-tumour potentials against cancerous basophils and breast cancer respectively

Possible implicationsResults and Discussion

14

Possible implications2) Modes of action of anti-tumour bioactive compounds

from vs31, vs38 and vs88 possibly specific and targeted

Since proliferation of cancerous basophils KU812 and breast cancer cells MB231 each inhibited by different microalgae strains

Interference only with oncogenic events pertaining to respective cancer cell line, not general proliferative mechanisms (e.g. disruption of DNA replication) (National Cancer Institute, 2014)

Results and Discussion

15

Test microalga with growth inhibitory effects, BY EXTRACTION SOLVENT

Hexane crude extract

Ethanol crude extract

Distilled water crude extract

Results and Discussion

16

Possible implications3) Anti-tumour bioactive compounds from vs38, vs88 and vs31

are likely of medium to high chemical polarity

12 out of 15 microalgae crude extracts inhibiting cancer growth were isolated using polar ethanol and distilled water extraction solvents

Extraction solvents “capture” compounds of like polarity

Results and Discussion

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37.1% 13.4%

30.4% 20.8% 34.2% 18.9%

Results and Discussion

Effects of test microalga on cancerous basophils KU812, BY GROWTH CONDITIONS

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Non-stressedStressed

Non-stressedStressed StressedNon-stressed

Results and Discussion

Effects of test microalga on breast cancer cell line MB231, BY GROWTH CONDITIONS

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Possible implications4) Bioactive compounds inhibiting KU812 and MB231 cancer

proliferation from vs31, vs38 and vs88 likely NOT lipids in nature

Non-stressed crude extracts should theoretically comprise smaller lipid content than stressed counterparts, due to lack of ultraviolet-C irradiation

Greater proportion of non-lipids stronger growth inhibition efficiencies

Results and Discussion

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Nature of microalgae crude extracts (inhibits or stimulates cancer cell growth) on KU812 cell line (purple) and MB231 cell line (blue)

Results and Discussion

Test microalga with growth stimulatory effects, BY GROWTH CONDITIONS

21

5) Lipids may play an assistive role in cancer cell proliferation

Crude extracts from stressed microalgae should theoretically comprise greater lipid content from ultraviolet-C irradiation

Greater abundance of lipids in stressed crude extracts translated to poorer cancer growth inhibition

Findings corroborate other literature reporting growth-stimulating effects of lipids on cancerous tumours (Baenke et al., 2013, Huang and Cheung., 2011)

Signalling molecules for cancer cell proliferation, migration, angiogenesis

Raw material for membrane synthesis and energy generation Unsaturated lipids increase cell membrane permeability and

nutrient intake

Possible implicationsResults and Discussion

22

Microscopic observationFigure 3.9

Anti-tumour effect determined at 0.25mg/mL extract exposure -> could be clearly observed using microscopy at 2mg/mL extract exposure

Results and Discussion

2mg/mL vs31 stressed Ethanol

2mg/mL KK6 stressed Ethanol

Complete medium only

0.1% (v/v) DMSO only [safe dose]

KU812

2mg/mL vs88 stressed dH2O

2mg/mL KK6 stressed dH2O

Complete medium only

0.1% (v/v) DMSO only [safe dose]

MB231

23

Anti-tumour test with Actinomycin-D The smaller the IC50 values, the greater the

potency of a particular compound or drug.

Gentler gradient= Wider therapeutic window=adverse drug event unlikely to be observed with subtle changes in drug concentration.

Steeper gradient= Narrower therapeutic windows adverse drug event likely to occur with subtle changes in drug concentration.

Results and Discussion

24

Anti-tumour test with Actinomycin-D

Figure 3.11

MB231 exposed to..

Gradient IC50 value

Stressed vs31 dH2O crude extract

-3.72 %.mg-1.mL 9.38 x 1010 mg/mL

Actinomycin-D -20.8 %.mg-1.mL 3.81mg/mL

Wider therapeutic window, Less potent

Results and Discussion

25

Anti-tumour test with Actinomycin-D

-D

-D

-D

-D

-D

-D

Non-stressed vs31 KU812 Hexane

Stressed vs31 KU812 Hexane

Non-stressed vs31 KU812 Ethanol

Non-stressed vs31 KU812 dH2O

Stressed vs31 KU812 Ethanol

Stressed vs31 KU812 dH2O

Figure 3.12

Results and Discussion

26

Gradient of Actinomycin-D > microalgae crude extract

-D

-D

-D

-D

-D

Non-stressed vs31 KU812 Hexane

Stressed vs31 KU812 Hexane

Non-stressed vs31 KU812 Ethanol

Stressed vs31 KU812 Ethanol

Stressed vs31 KU812 dH2O

Figure 3.12

KU812 exposed to..

Gradient IC50 value

Non-stressed vs31 Hexane crude extract

-8.45 %.mg-1.mL

0.554mg/mL

Non-stressed vs31 Ethanol crude extract

-9.11%.mg-1.mL

93.8mg/mL

Stressed vs31 dH2O crude extract

-2.46%.mg-1.mL

1.00 x 1011mg/mL

Actinomycin-D

-14.8 %.mg-1.mL

13.9mg/mL

Narrowest therapeutic window

Most potent

Results and Discussion

27

Gradient of Actinomycin-D < microalgae crude extract

-D

-D

Stressed vs31 KU812 Hexane

Stressed vs31 KU812 Ethanol

Figure 3.12

KU812 exposed to..

Gradient IC50 value

Stressed vs31 Hexane crude extract

-17.4 %.mg-1.mL

22.5mg/mL

Stressed vs31 Ethanol crude extract

-44.1%.mg-1.mL

0.812mg/mL

Actinomycin-D

-14.8 %.mg-1.mL

13.9mg/mL

Most potentNarrowest therapeutic window

Results and Discussion

28

Anti-tumour test with Actinomycin-D

Non-stressed vs31 KU812 dH2O

KU812 exposed to..

Gradient IC50 value

Non-stressed vs31 dH2O crude extract

+16.9 %.mg-1.mL

3.35 x 10-2mg/mL

Actinomycin-D

-14.8 %.mg-1.mL

13.9mg/mL

Figure 3.12

• At 0.25mg/mL of the extract significantly depicted that it inhibited the proliferation of KU812

• However, a positive correlation observed between cell viability & concentration of the extract

• High amount of bioactive compounds which had promoted cellular proliferation

Results and Discussion

-D

29

Anti-tumour test with Actinomycin-D

-D

-D

-D

-D

-D

-D

Non-stressed vs31 KU812 Hexane

Stressed vs31 KU812 Hexane

Non-stressed vs31 KU812 Ethanol

Non-stressed vs31 KU812 dH2O

Stressed vs31 KU812 Ethanol

Stressed vs31 KU812 dH2O

Figure 3.12

• 50% tested microalgae extracts on KU812 cell line (B, C and F) displayed higher IC50 values than Actinomycin-D Crude Concentration of anti-tumour metabolites smaller

per unit volume

• Other 50% of the tested microalgae extracts on KU812 cell line (A, D and E) revealed smaller IC50 values than Actinomycin-D Crude High concentration of anti-tumour metabolites

OR Small concentration of anti-tumour metabolites had

targeted a critical apoptotic cascade-> greater effect in inhibiting KU812 growth

Results and Discussion

30

• Identity of components exhibiting growth inhibitory effects could have been conclusively verified using chromatography

• Prospective use of microalgae crude extracts as anti-tumour therapy in humans could have been substantiated by conducting cytotoxicity tests on non-transformed human cell lines

• Examining local microalgae species for anti-oxidant properties and potential to repress malignant transformation and cancer onset

Future Work

31

Summary

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Investigating anti-tumour effects from microalgae isolated from Singapore’s water bodies

By Strain

KK6 (NIL)

vs31 inhibited basophil leukemic cells

vs38 & vs88 inhibited breast cancer cells

By Extraction Solvent

12/15 are ethanol & dH2O

Anti-tumour bioactive compounds = medium to high chemical polarity, with targeted modes of action

3/15 are hexane

Lipids

By Growth Condns

Stressed

Non-stressed

Lipids

Side Proje

ctVarying potencies & therapeutic windows as compared to those of Actinomycin-D

Possible clinical treatment of cancer with local

microalgae strains in future

Thank you.

33

Baenke, F., Peck, B., Miess, H., and Schulze, A., 2013. Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development. Disease models and mechanisms, 6(6), pp. 1353-1363.

Boopathy, N. S., and Kathiresan, K., 2010. Anticancer Drugs from Marine Flora: An Overview. Journal of Oncology, Volume 2010, pp. 1-18.

Huang, J.-h. J., and Cheung, C.-K. P., 2011. +UVA treatment increases the degree of unsaturation in microalgal fatty acids and total carotenoid content in Nitzchia closterium (Bacillariophyceae) ad Isochrysis zhangjiangensis (Chrysophyceae). Food Chemistry, Volume 129, pp. 783-791.

Ibrahim, A. M. M., Mostafa, M. H., El-Masry, M. H. and El-Naggar, M. M. A., 2005. Active biological materials inhibiting tumour initiation extracted from marine algae. Egyptian Journal of Aquatic Research, Volume 31(1). pp. 146-155.

Lin, H. C., Chou, A. T., Chuang, M. Y., Liao, T. Y., Tsai, W. S., and Chiu, T. H., 2012. The effects of Caulerpa microphysa enzyme-digested extracts on ACE-inhibitory activity and in vitro anti-tumour properties. Food Chemistry, Volume 134. pp. 2235-2241.

Mujtaba, G., Choi, W., Lee, C.-G., and Lee, K., 2012. Lipid production by Chlorella vulgaris after a shift from nutrient-rich to nitrogen starvation conditions. Bioresource Technology, Volume 123, pp. 279-283.

National Cancer Institute, 2014. Targeted Cancer Therapies. [Online] Available at: http://www.cancer.gov/cancertopics/factsheet/Therapy/targeted[Accessed 12 December 2014]. Singh, S., Kate, B. N., and Banerjee, U. C., 2005. Bioactive Compounds from Cyanobacteria and microalgae: An Overview. Critical Reviews in Biotechnology, Volume 25, pp. 73–95

Bibliography

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Cultivation & harvesting of microalgae strains Mass of dried extracts were recorded

Re-dissolve dried extracts in DMSO to achieve a Master stock (2000mg/mL) before serial dilutions to produce 4 more Master stocks:

2000mg/mL

1000mg/mL

500mg/mL

250mg/mL 62.5mg/mL

Materials and Methods

Microtube

Glass tube

35

Microscopic observation

KU812 Comparison

MB231 Comparison

A B

G H

C D E

I J K

Figure 3.9

• Cells were viewed under 400x magnification using Olympus CK40 inverted microscopy

• Comparison of the cell morphology/cell confluency & presence of dark blue precipitate inside cells after incubation with MTT for~3hours

• C and I are negative controls (exposed to complete medium only)

• D and J are “Safe-DMSO” controls

• E and K are “Lethal-DMSO” controls

Effectiveness of microalgae extracts on cancer cells?

Cell type Effective IneffectiveKU812 A B

2mg/mL vs31 stressed Ethanol

2mg/mL KK6 stressed Ethanol

MB231 G H2mg/mL vs31 stressed dH2O

2mg/mL KK6 stressed dH2O

• Anti-tumour effect determined at 0.25mg/mL extract exposure -> could be clearly observed using microscopy at 2mg/mL extract exposure

Results and Discussion

36

Microalgae strains vs31, vs38 and vs88 displayed anti-cancer properties, except for KK6

Anti- tumour bioactive compounds likely of medium to high chemical polarity, with targeted modes of action

Anti-tumour bioactive compounds unlikely to be lipids-based

The peculiar case of non-stressed vs31 distilled water against KU812 (Figure 3.12E)

Varying potencies & therapeutic windows as compared to

those of Actinomycin-D

Possible clinical treatment of cancer with local microalgae strains in future

Conclusions

37